Methods and compositions for in vitro synthesis of biological macromolecules in a cell-free system enriched with atp-sulfurylase

Abstract

The present invention provides methods and composition for enhancing in vitro synthesis of biological macro-molecules in a call-free system where ATP is required as a primary energy source, wherein said cell-free system is enriched with ATP-sulfurylase The invention relates also to as cell-free systems and cell-free extracts enriched with ATP-sulfurylase for enhancing in vitro synthesis of any biological macromolecules.

1 . A method for enhancing in vitro synthesis of proteins and fragments thereof in a cell-free system comprising endogenous adenosine 5′ phosphosulfate where ATP is required as a primary energy source, comprising enriching said cell-free system with ATP-sulfurylase.  
     
     
         2 . A method according to  claim 1 , wherein the cell free system further comprises exogenous adenosine 5′ phosphosulfate.  
     
     
         3 . The method according to  claim 1 , wherein said in vitro synthesis also comprises transcription of mRNA from a DNA template.  
     
     
         4 . A method according to  claim 1 , comprising carrying out said in vitro synthesis in a reaction vessel as a batch reaction, semi continuously or continuously.  
     
     
         5 . A method according to  claim 1 , comprising adding ATP-sulfurylase to the cell-free system at the beginning and/or during the in vitro synthesis or at intervals during the in vitro synthesis.  
     
     
         6 . A method according to  claim 1 , wherein the cell-free system comprises a cell-free extract prepared from cells transformed with a vector over-expressing ATP-sulfurylase.  
     
     
         7 . A method according to  claim 1 , comprising adapting ATP-sulfurylase concentration according to the experimental conditions and the biological macromolecules to be synthesized.  
     
     
         8 . A method according to  claim 1 , wherein ATP-sulfurylase is present in the cell-free system at an initial concentration of at least about 0.1 U/ml.  
     
     
         9 . A cell-free system comprising components that are capable of translating messenger ribonucleic acid encoding a desired protein enriched with ATP-sulfurylase.  
     
     
         10 . A cell-free system according to  claim 9  comprising exogenous adenosine 5′ phosphosulfate.  
     
     
         11 . A cell-free system according to  claim 9  comprising all substances necessary for the translation of mRNA and transcription of mRNA from a DNA template.  
     
     
         12 . A cell-free system according to  claim 9 , wherein extra ATP-sulfurylase is derived from a prokaryotic organism, an eukaryotic organism, a transgenic vector, a bacterial cell that has been genetically modified,  E. coli  extract, or is purified.  
     
     
         13 . A cell-free system according to  claim 9 , wherein the cell-free extract enriched with ATP-sulfurylase is prepared from cells transformed with a vector over-expressing ATP-sulfurylase.  
     
     
         14 . A cell-free system according to  claim 9 , wherein ATP-sulfurylase is present in a concentration of at least about 0.1 U/ml.  
     
     
         15 . A cell-free extract comprising components that are capable of translating messenger ribonucleic acid encoding a desired protein enriched with ATP-sulfurylase.  
     
     
         16 . A cell-free extract according to  claim 15  comprising exogenous adenosine 5′ phosphosulfate.  
     
     
         17 . A cell-free extract according to  claim 15  comprising all substances necessary for the translation of mRNA and transcription of mRNA from a DNA template.  
     
     
         18 . A cell-free extract according to  claim 15 , wherein extra ATP-sulfurylase is derived from a prokaryotic organism, a eukaryotic organism, a transgenic vector, a bacterial cell that has been genetically modified, an  E. coli  extract, or is purified.  
     
     
         19 . A cell-free extract according to  claim 15  prepared from cells transformed with a vector over-expressing ATP-sulfurylase.  
     
     
         20 . A cell-free extract according to  claim 15 , wherein ATP-sulfurylase is present in a concentration of at least about 0.1 U/ml.  
     
     
         21 . A method for enhancing in vitro synthesis of polypeptides, comprising: 
 (a) providing a cell-free system comprising mRNA and adenosine 5′ phosphosulfate and enriched with ATP-sulfurylase; and    (b) translating said mRNA.    
     
     
         22 . A cell-free system for mRNA translation comprising components for cell-free mRNA translation, wherein said system is enriched with ATP-sulfurylase.